Back to EveryPatent.com
| United States Patent |
5,256,551
|
|
Terasawa
, et al.
|
October 26, 1993
|
Method of treating microorganism cells containing tryptophanase or
treated product thereof
Abstract
A method of selectively inhibiting pyruvic acid decomposition activity in
microorganism cells containing tryptophanase or a treated product thereof,
which comprises heat-treating said cells or the treated product thereof in
the presence of an ammonium ion.
| Inventors:
|
Terasawa; Masato (Ami, JP);
Shimazu; Mitsunobu (Ami, JP);
Endo; Fuzio (Ami, JP);
Yukawa; Hideaki (Ami, JP)
|
| Assignee:
|
Research Association for Utilization of Light Oil (Tokyo, JP)
|
| Appl. No.:
|
731309 |
| Filed:
|
July 17, 1991 |
Foreign Application Priority Data
| Current U.S. Class: |
435/108; 435/252.8; 435/260; 435/849 |
| Intern'l Class: |
C12P 013/22; C12N 001/04; C12N 001/20 |
| Field of Search: |
435/108,849,260,252.8
|
References Cited
| Foreign Patent Documents |
| 045307 | Mar., 1980 | JP | 435/108.
|
| 134094 | Jun., 1987 | JP | 435/108.
|
| 883029 | Nov., 1981 | SU | 435/108.
|
Other References
"Patent Abstracts of Japan", unexamined applications, C field, vol. 12, No.
391, Oct. 18, 1988, p. 83 C 537.
"Patent Abstracts of Japan", unexamined applications, C field, vol. 11, No.
365, Nov. 27, 1987, p. 1 C 460.
|
Primary Examiner: Marx; Irene
Attorney, Agent or Firm: Wenderoth, Lind & Ponack
Parent Case Text
This application is a continuation of now abandoned application, Ser. No.
07/177,945 filed Apr. 5, 1988.
Claims
What we claim is:
1. A method of selectively inhibiting pyruvic acid decomposition activity
in Escherichia coli selected from the group consisting of Escherichia coli
K-12 YK3002, Escherichia coli YK3003, Escherichia coli ATCC 27325,
Escherichia coli ATCC 25019 and Escherichia coli IFO 3301, which comprises
heat-treating said Escherichia coli at a temperature of 50.degree. to
70.degree. C. in the presence of ammonium ion of a concentration of 100 to
10,000 mg/liter.
2. The method of claim 1 wherein ammonium chloride, ammonium sulfate or
ammonium phosphate is used as a source of the ammonium ion.
3. The method of claim 1 wherein the heat-treatment is carried out in an
aqueous medium.
4. The method of claim 1 wherein the concentration of the ammonium ion is
500 to 8,000 mg/liter.
5. The method of claim 3 wherein the pH of the aqueous medium at the time
of the heat-treatment is 5 to 9.
6. The method of claim 5 wherein the pH of the aqueous medium at the time
of the heat-treatment is 6 to 8.
7. The method of claim 3 wherein the concentration of the cells or the
treated product thereof in the aqueous medium is 0.1 to 50% (wt/vol).
8. The method of claim 1 in which the heat treatment is carried out at a
temperature of 55.degree. to 60.degree. C.
9. The method of claim 1 wherein said Escherichia coli is in the form of
(1) cells of said Escherichia coli, (2) ruptured cells of said Escherichia
coli, (3) cell free extracts of said Escherichia coli or (4) immobilized
preparation thereof.
10. A method of producing L-tryptophan by reacting indole, pyruvic acid or
its salt, and ammonia or ammonium ion in the presence of Escherichia coli
selected from the group consisting of Escherichia coli K-12 YK-3002,
Escherichia coli K-12 YK3003, Escherichia coli ATCC 27325, Escherichia
coli ATCC 25019 and Escherichia coli IFO 3301, and harvesting L-tryptophan
from the reaction mixture, wherein said Escherichia coli has been
subjected to heat-treatment at a temperature of 50.degree. to 70.degree.
C. in the presence of ammonium ion at a concentration of 100 to 10,000
mg/liter.
11. The method of claim 10 wherein said Escherichia coli is in the form of
(1) cells of said Escherichia coli, (2) ruptured cells of said Escherichia
coli, (3) cell free extracts of said Escherichia coli or (4) immobilized
preparation thereof.
Description
This invention relates to a method of treating microorganism cells
containing tryptophanase or a treated product thereof. More specifically,
this invention relates to a method of selectively inhibiting the
decomposition activity of pyruvic acid without substantially decreasing
tryptophanase activity in microorganism cells containing tryptophanase or
a treated product thereof.
It is known that L-tryptophan is produced by enzymatically reacting indole,
pyruvic acid or its salts, and ammonium or an ammonium ion in the presence
of microorganism cells containing tryptophanase or a treated product
thereof [T. Watanabe and E. E. Snell, "Proceedings of National Academy of
Science, U. S. A.", vol. 69, No. 5, pages 1086-1090 (1972)].
Pyruvic acid is one substance which participates in various metabolisms in
animals and plants, and microorganism cells containing tryptophanase or a
treated product thereof contains an enzyme which is involved in the
metabolism of pyruvic acid in addition to tryptophanase. If, therefore,
the microorganism cells containing tryptophanase or the treated product
thereof is used in the production of L-tryptophan by enzymatic reaction,
the starting pyruvic acid or its salt is used in the formation of
L-tryptophan and also consumed by the decomposition reaction of pyruvic
acid. Hence, the yield of the desired L-tryptophan decreases.
The freezing-thawing method and the heat-treatment method are generally
known for the inhibition of enzyme activity. If these methods are applied
to the microorganism cells containing tryptophanase or the treated product
thereof, not only the pyruvic acid decomposition reaction but also the
tryptophanase activity is inhibited. Accordingly, these methods cannot be
used.
The present inventors made extensive investigations on a method of
inhibiting the pyruvic acid decomposition activity of an enzyme
participating in the metabolism of pyruvic acid without substantially
reducing the tryptophanase activity in microorganism cells containing
tryptophanase or the treated product thereof. These investigations have
led to the discovery that when the microorganism cells containing
tryptophanase or the treated product thereof is heat-treated in the
presence of an ammonium ion, the pyruvic acid decomposition activity can
be selectively inhibited without substantially reducing the tryptophanase
activity.
According to this invention, there is provided a method of selectively
inhibiting pyruvic acid decomposition activity in microorganism cells
containing tryptophanase or a treated product thereof, which comprises
heat-treating said microorganism cells or the treated product thereof in
the presence of an ammonium ion.
If the microorganism cells or the treated product thereof, which is
heat-treated by the method of this invention, is used for the production
of L-tryptophan from indole, pyruvic acid or its salt and ammonia or an
ammonium ion, the consumption of pyruvic acid by a side-reaction is
inhibited, the conversion of pyruvic acid into L-tryptophan is effectively
promoted, and the yield of L-tryptophan can be markedly increased.
The tryptophanase-containing cells used in the method of this invention
include cells containing tryptophanase produced and accumulated
sufficiently therein as a result of cultivating microorganisms having the
ability to produce tryptophanase. Such microorganisms may be of any family
or genus if they have the ability of producing tryptophanase. Specific
examples are given below. It should be understood however that they are
mere examples, and the scope of the invention is not limited thereto.
Escherichia coli ATCC 27325
Escherichia coli ATCC 25019
Escherichia coli IFO 3301
Escherichia coli K-12 YK3002 (FERM BP-1733)
Escherichia coli K-12 YK3003 (FERM BP-1734)
Providencia rettgeri ATCC 9250
Proteus vulgaris ATCC 6059
Flavobacterium meningosepticum ATCC 13253
These tryptophanase-producing microorganisms may be used in the method of
this invention after they are cultured to accumulate tryptophanase
sufficiently within their cells. Culturing of the microorganisms may
generally be carried out by using ordinary synthetic or natural media
containing carbon sources, nitrogen sources and inorganic substances. The
carbon sources may be, for example, various carbohydrates such as glycose,
glycerol, fructose, sucrose and molasses. The nitrogen sources include,
for example, natural organic nitrogen sources such as tryptone, yeast
extract, corn steep liquor, and casein hydrolyzate. Many of the natural
organic nitrogen sources can also serve as carbon sources. Further
examples of nitrogen sources are ammonium salts such as ammonium chloride,
ammonium sulfate and ammonium phosphate, nitrates such as sodium nitrate,
potassium nitrate and ammonium nitrate, and ammonia. Examples of the
inorganic substances are potassium phosphate, magnesium sulfate, iron,
manganese and zinc. Examples of the inorganic substances are potassium
phosphate, magnesium sulfate, iron, manganese and zinc. As required,
nutrients such as vitamins and amino acids may be added.
The culturing may be carried out under aerobic conditions by shaking
culture, or submerged culture with aeration and agitation. The culturing
temperature is generally 20.degree. to 50.degree. C., preferably
30.degree. to 40.degree. C. Desirably, the pH of the culture medium is
usually 6 to 9, preferably 7 to 8. The culturing period is usually 1 to 5
days.
If an iron ion is continuously or intermittently added to the culture
medium so that the iron ion concentration is maintained at not more than
0.15 mM, preferably 0.02 to 0.1 mM, the microorganism having a high
tryptophanase activity can be obtained in a large quantity. As a source of
the iron ion to be added to the medium, there may be used, for example,
iron sulfate, iron chloride and iron nitrate. These salts may be added to
the culture medium while the concentration of the iron ion is monitored in
a conventional manner by, for example, colorimetry.
Microorganism cells having high tryptophanase activity can also be obtained
in large quantities by continuously or intermittently adding an alkaline
substance to the culture medium so as to maintain the pH of the culture
medium at 7.0 to 7.4. The alkaline substance that can be used for pH
control of the medium may be a substance which when added to the culture
medium, causes the pH of the culture medium to shift to an alkaline side,
and does not substantially exert a deleterious action on the microorganism
in the culture medium. Examples may be ammonia, aqueous ammonia, alkalies
and aqueous solutions of alkalies. Aqueous ammonia, aqueous solution of
sodium hydroxide, an aqueous solution of potassium hydroxide and an
aqueous solution of calcium hydroxide are preferred.
The tryptophanase-producing cells so cultured are harvested from the
culture medium, and may be heat-treated by the method of this invention
either directly or after the harvested microorganism cells are treated by
physical means such as ultrasonication and squeezing to rupture the cell
membranes; by chemical means using chemicals or enzymes to rupture the
cell membranes; extracts obtained by extracting these ruptured products
with water or another extractant; or a "treated product" of the above
tryptophanase-containing cells obtained by treating the extracts with
ammonium sulfate or the like to precipitate the enzyme component. Products
obtained by immobilizing the above cells or the treated product thereof,
by a known method, for example the method described in "Methods in
Enzymology", vol. 44 (1977), edited by K. Moskauch, may be used as the
"treated product" of the cells in the method of this invention.
According to the method of this invention, the tryptophanase-containing
cells or the treated product thereof is heat-treated in the presence of an
ammonium ion. This heat-treatment may be carried out by suspending the
cells or the treated product thereof in an aqueous medium and adding an
ammonium ion source. The ammonium ion source that can be added may be any
of ammonium-containing compounds which dissolve in water to liberate an
ammonium ion and do not contain components that substantially adversely
affect the tryptophanase activity. Examples of preferred
ammonium-containing compounds are ammonium chloride, ammonium sulfate and
ammonium phosphate. Ammonium nitrate should not be used because it
inhibits tryptophanase.
The concentration of the ammonium ion in the aqueous medium is not strictly
limited, and may be varied over a wide range according to the type of the
cells or the treated product thereof, and the degree of treatment.
Generally, it is 100 to 10,000 mg/liter, preferably 500 to 8,000 mg/liter,
especially preferably 1,000 to 5,000 mg/liter, as the ammonium (NH4) ion.
The pH of the aqueous medium used in the heat-treatment is generally 5 to
9, preferably 6 to 8, more preferably 7 to 8.
Water or a buffer solution such as potassium phosphate buffer or
tris-hydrochloric buffer may be generally used as the aqueous medium. If
desired, a minor proportion of a water-miscible surface-active agent,
preferably a nonionic surface-active agent such as Triton X-100, Tween 20
and Tween 40, may be added to the medium.
The concentration of the cells or the treated product thereof in the
aqueous medium is neither critical, and may be varied widely depending
upon the concentration of the ammonium ion. Generally, it is 0.1 to 50 %
(wt/vol), preferably 0.5 to 30 % (wt/vol), more preferably 1 to 20 %
(wt/vol). When the concentration of the cells or the treated product
thereof is high, the concentration of the ammonium ion is desirably higher
within the above range. Any one skilled in the art would be able to
determine the optimum combination of these concentrations by performing a
small-scale routine experiment.
The heat-treating temperature in accordance with this invention is
generally 40.degree. to 80.degree. C., preferably 50.degree. to 70.degree.
C., especially preferably 55.degree. to 60.degree. C. At this temperature,
the heat-treatment may be carried out usually for 2 minutes to 24 hours,
preferably about 5 minutes to 6 hours.
The tryptophanase-containing cells or the treated product thereof, which
has been heat-treated as above by the method of this invention, has
markedly inhibited pyruvic acid decomposition activity without a
substantial reduction in tryptophanase activity, and can advantageously be
used as an enzyme catalyst in the production of L-tryptophan by the
reaction of indole, pyruvic acid or its salt and ammonia or an ammonium
ion, as will be demonstrated by examples given below.
The reaction of synthesizing L-tryptophan can be carried out in quite the
same way as in known L-tryptophan-synthesizing reactions except that the
tryptophanase-containing cells or the treated product thereof, which is
heat-treated by the method of the invention, is used. For example, it is
carried out in a solvent such as 0.1 M phosphate buffer (pH 7.5-10.0) or
water (pH 7.5-10.0) at a temperature of about 20.degree. to 50.degree. C.,
preferably about 30.degree. to 40.degree. C., for a period of usually
about 2 to 72 hours.
There is no particular restriction on the amounts of indole, pyruvic acid
or its salt (such as an alkali metal pyruvate), and ammonium or an
ammonium ion (an ammonium salt of an organic acid such as ammonium acetate
may also be used besides the above-mentioned ammonium ion sources) used in
the reaction so long as they are used in concentrations which do not
inhibit the enzymatic reaction. Generally, the suitable concentration of
each of these materials is in the range of 0.1 to 20% (wt/vol), preferably
0.5 to 10% (wt/vol). The ratio of indole/pyruvic acid or its salt/ammonia
or the ammonium ion is neither restricted strictly. Generally, the mole
ratio of these is suitably 1:1-100:1-300, preferably 1:5-50:5-100.
The amount of the cells or the product thereof is not particularly
restricted. Generally, they may be used in a concentration of 0.5 to 10%
(wt/vol), preferably 0.8 to 8% (wt/vol).
Isolation of L-tryptophan from the reaction mixture after the reaction and
its purification may be carried out by known methods, for example,
adsorption and desorption on and from ion exchange resins and activated
carbon.
The following Examples illustrate the present invention in greater detail.
EXAMPLE 1
One hundred milliliters of a an L-medium composed of 10 g of tryptone, 5 g
of yeast extract, 5 g of sodium chloride, 1 g of glucose and 1 liter of
distilled water (pH 7.2) was put in a 500 ml Erlenmeyer flask, and
sterilized at 120.degree. C. for 15 minutes. Escherichia coli K-12 YK3003
(FERM BP-1733), a tryptophanase-producing microorganism, was inoculated in
the L-medium, and cultured at 37.degree. C. for 1 day under shaking. Then,
20 ml of the culture broth was inoculated in 1,000 ml of L-medium
containing L-tryptophan in a concentration of 200.mu.g/ml (sterilized at
120.degree. C. for 15 minutes) and cultured at 37.degree. C. under
shaking. The cells were harvested by centrifugation (6000 rpm, 15 minutes,
4.degree. C.) from 100 ml of the resulting culture broth. The harvested
cells were suspended in 10 ml of distilled water containing 5 % by weight
of ammonium sulfate. The cell suspension was heat-treated for each of the
periods of time indicated in Table 1, and then pyruvic acid decomposition
activity and tryptophanase activity were measured.
The pyruvic acid decomposition activity was measured by the following
procedure. All the treated cells were added to 25 ml of a reaction
solution composed of 200 .mu.mole of sodium pyruvate, 0.04 .mu.mole of
pyridoxalphosphoric acid, 100 .mu.mole of NH4Cl, and distilled water to
make 1 ml (pH 8.7 adjusted with 5N sodium hydroxide) and reacted at
37.degree. C. for 24 hours. The amount of pyruvic acid was measured by
liquid chromatography.
The tryptophanase activity was measured by the following procedure.
0.1 ml of the aforesaid treated cell suspension was added to 25 ml of a
reaction solution which contained per ml thereof, 100.mu.mole of phosphate
buffer (pH 8.0), 5 .mu.mole of L-tryptophan and 0.04.mu.mole of
pyridoxalphosphoric acid, and reacted at 37.degree. C. for 15 minutes. The
amount of indole formed was determined by a conventional method [O. H.
Smith and C. Janofsky: "Methods in Enzymology", Academic, New York, (1962,
vol. 5, pages 794-806)].
The pyruvic acid decomposition activity and the tryptophanase activity are
shown in Table 1 as relative activities which were obtained by taking the
enzyme activity before heat-treatment as 100.
TABLE 1
______________________________________
Treating conditions
Pyruvic acid
Trypto-
Temper- decomposition
phanase
ature Time activity activity
(.degree.C.)
(min.) (%) (%)
______________________________________
-- 0 100 100
(before heat-
treatment)
45 60 50 99
90 42 99
50 30 35 99
60 26 98
55 15 21 95
30 10 94
60 10 9 91
20 7 86
65 5 9 88
10 8 70
20 6 62
70 5 5 41
______________________________________
EXAMPLE 2
The same microorganism as used in Example 1 was cultured under shaking as
in Example 1, and further cultured under shaking in L-medium containing
L-tryptophan. 25 ml of the resulting culture broth was centrifuged (6000
rpm, 15 minutes, 4.degree. C.) to harvest the cells. The resulting cells
were washed once with 20 ml of 100 mM Tris-HCl buffer (pH 7.5) and
suspended in 1 ml of the same buffer. The cell suspension was subjected to
rupturing by a cell rupturing device (Branson S-200). The ruptured product
was centrifuged (12000 rpm, 40 minutes, 4.degree. C.) to separate it into
a supernatant and a precipitate.
Ammonium sulfate was added to 1 ml of the above supernatant (cell-free
extract), and the concentration of ammonium sulfate was adjusted to 5 % by
weight. The solution was heat-treated for each of the periods of time
indicated in Table 2. The pyruvic acid decomposition activity and the
tryptophanase activity were measured.
The pyruvic acid decomposition activity was measured by the following
procedure. 0.1 ml of the heat-treated product was added to 1 ml of a
reaction solution composed of 200 .mu.moles of sodium pyruvate, 0.04
.mu.mole of pyridoxalphosphoric acid, 100 .mu.mole of NH.sub.4 Cl and
distilled water to make 1 ml (pH 80 adjusted with 5N sodium hydroxide) and
reacted at 37.degree. C. for 24 hours. The amount of the remaining pyruvic
acid was measured by liquid chromatography.
The tryptophanase activity was measured by the following procedure. 0.1 ml
of a diluted solution of the heat-treated product (diluted to 100-fold
with 100 mM Tris-HCl buffer, pH 8.0) was added to 1 ml of a reaction
solution containing 100 .mu.mole of phosphate buffer (pH 8.0), 5 .mu.mole
of L-tryptophan and 0.04 .mu.mole of pyridoxalphosphoric acid, and reacted
at 37.degree. C. for 15 minutes. Thereafter, the tryptophanase activity
was measured by the same method as in Example 1.
The results are shown by relative activity values as in Example 1 in Tale
2.
TABLE 2
______________________________________
Treating conditions
Pyruvic acid
Trypto-
Temper- decomposition
phanase
ature Time activity activity
(.degree.C.)
(min.) (%) (%)
______________________________________
-- 0 100 100
(before
heat-treatment)
40 60 62 98
90 48 98
50 20 28 98
30 15 97
55 10 6 97
20 3 95
60 5 3 91
10 2 84
______________________________________
EXAMPLE 3
Example 1 was repeated except that in the heat-treatment, the temperature
was changed to 60.degree. C. and the time, to 10 minutes, and the
concentration of the ammonium ion was changed as shown in Table 3. The
results are shown in Table 3.
TABLE 3
______________________________________
Concentration Trypto- Pyruvic acid
of the ammonium
phanase decomposition
ion added activity activity
(wt %) (%) (%)
______________________________________
as (NH.sub.4).sub.2 SO.sub.4
0 45 45
0.1 68 20
0.5 75 16
1 88 12
5 91 9
10 91 9
20 92 8
as NH.sub.4 Cl
0 45 45
0.2 73 15
0.4 87 10
4 90 9
8 91 9
as (NH.sub.4).sub.3 PO.sub.4
0 45 46
0.15 69 22
1.5 87 11
15 90 8
______________________________________
EXAMPLE 4
Example 2 was repeated except that in the heat-treatment, the temperature
was changed to 55.degree. C. and the time, to 10 minutes, and the
concentration of the ammonium ion was changed as shown in Table 4. The
results are shown in Table 4.
TABLE 4
______________________________________
Concentration Trypto- Pyruvic acid
of the ammonium
phanase decomposition
ion added activity activity
(wt %) (%) (%)
______________________________________
as (NH.sub.4).sub.2 SO.sub.4
0 43 40
0.1 75 14
0.5 90 8
1 94 8
5 97 6
10 97 6
20 97 6
as NH.sub.4 Cl
0 44 39
0.2 91 7
0.4 93 7
4 96 6
8 97 6
as (NH.sub.4).sub.3 PO.sub.4
0 43 40
0.15 74 15
1.5 94 8
15 97 6
______________________________________
EXAMPLE 5
1000 ml of a culture broth prepared in the same way as in Example 1 was
centrifuged (6000 rpm, 15 minutes, 4.degree. C.) to harvest the cells. The
cells were suspended in 100 ml of distilled water containing 50 mg/ml of
ammonium sulfate. The cell suspension was heat-treated in a
constant-temperature vessel at 60.degree. C. for 10 minutes with shaking,
and then centrifuged (6000 rpm, 15 minutes, 4.degree. C.) to harvest the
cells. The cells were then suspended in 50 ml of 100 mM Tris-Hcl buffer
containing 0.25 g of indole, 0.25 g of sodium pyruvate, 0.5 g of ammonium
acetate, 0.5 mg of pyridoxalphosphoric acid and 0.5 g of KCl, and with
shaking, these compounds were reacted at 37.degree. C. for 3 hours. After
the reaction, the reaction mixture was diluted to 10-fold with water, and
centrifuged. The supernatant was analyzed for L-tryptophan by
high-performance liquid chromatography. The formation of 0.62 m9/ml of
L-tryptophan was determined. After the reaction, 500 ml of the aforesaid
10-fold dilution was passed through a column of strong acid type
ion-exchange resin (ammonia type; "DIAION.RTM. SK-1B", a product of
Mitsubishi Chemical Co., Ltd.). The column was eluted with an alkaline
solution, and the eluate was concentrated to precipitate crude crystals of
L-tryptophan. The crude crystals were washed with acetone and dried to
give 205 mg of crystals of L-tryptophan.
When the above reaction was repeated using the aforesaid cells without
heat-treatment, the amount of L-tryptophan formed was 0.41 mg/ml.
EXAMPLE 6
Example 5 was repeated except that Escherichia coli K-12 YK3003 (FERM
BP-1734) was used instead of the microorganism used in Example 5.
L-tryptophan formed in an amount of 0.63 mg/ml when the heat-treated cells
were used, and in an amount of 0.39 mg/ml when the non-heat-treated cells
were used.
The amount of L-tryptophan crystals recovered from 50 ml of the reaction
mixture obtained by the reaction carried out by using the heat-treated
cells was 208 mg.
Top